Professional Documents
Culture Documents
Protein Tyrosine Phosphatase 1B Restrains Mammary Alveologenesis and Secretory Differentiation
Protein Tyrosine Phosphatase 1B Restrains Mammary Alveologenesis and Secretory Differentiation
SUMMARY
Tyrosine phosphorylation plays a fundamental role in mammary gland development. However, the role of specific tyrosine
phosphatases in controlling mammary cell fate remains ill defined. We have identified protein tyrosine phosphatase 1B (PTP1B) as
an essential regulator of alveologenesis and lactogenesis. PTP1B depletion increased the number of luminal mammary progenitors
in nulliparous mice, leading to enhanced alveoli formation upon pregnancy. Mechanistically, Ptp1b deletion enhanced the expression
of progesterone receptor and phosphorylation of Stat5, two key regulators of alveologenesis. Furthermore, glands from Ptp1b
knockout mice exhibited increased expression of milk proteins during pregnancy due to enhanced Stat5 activation. These findings
reveal that PTP1B constrains the number of mammary progenitors and thus prevents inappropriate onset of alveologenesis in early
pregnancy. Moreover, PTP1B restrains the expression of milk proteins during pregnancy and thus prevents premature lactogenesis.
Our work has implications for breast tumorigenesis because Ptp1b deletion has been shown to prevent or delay the onset of
mammary tumors.
KEY WORDS: PTP1B (Ptpn1), Stat5, Mammary gland, Stem cell, Progenitor cell, Mouse
progenitor cells in nulliparous mice, induces precocious Membranes were blocked in PBS with 5% skimmed milk powder and
formation of alveoli, and enhances the expression of milk incubated with PTP1B (Klaman et al., 2000), pStat5 (Cell Signaling) and
proteins during pregnancy. Stat5a (Transduction Laboratories) antibodies. Antibody binding was
visualized by incubation of secondary antibodies comprising Alexa
MATERIALS AND METHODS Fluor 680 anti-mouse IgG, Alexa Fluor 680 anti-rabbit IgG (Molecular
Mice Probes, Invitrogen), IRDye 800 anti-mouse IgG and IRDye 800 anti-
All animal experiments were performed according to Swiss guidelines rabbit IgG (Rockland), and examined with an Odyssey infrared imaging
governing animal experimentation and were approved by the Swiss system (Li-Cor Bioscience).
veterinary authorities. Ptp1b–/– mice (Klaman et al., 2000) were
backcrossed to an FVB background for at least seven generations. Twelve- Real-time PCR
week-old female mice were mated and pregnancy scored by the Total RNA was isolated from frozen mammary glands using TRIzol
observation of a vaginal plug and confirmed by the presence of fertilized reagent (Invitrogen) according to the manufacturer’s instructions and then
eggs or embryos when mammary glands were collected at pregnancy days treated with the TURBO-DNase Kit (Applied Biosystems). cDNA
3, 7 or 10. Mammary glands from nulliparous mice were collected when synthesis was performed using the Thermoscript RT-PCR system
mice were in estrus, as determined by a vaginal plug after an overnight (Invitrogen). Real-time PCR was performed on 30-60 ng cDNA using the
mating with a male. TaqMan Gene Expression Assay (Applied Biosystems) for Wap
(Mm00839913_m1), -casein (Mm00839664_m1), cytokeratin 18
Whole-mounts and histological analysis (Mm01601702_g1), Rankl (Mm00441906_m1), Pr (Mm00435628_m1),
For whole-mounts and histology, inguinal and thoracic mammary glands Er (Mm00433149_m1) and Gapdh (Rodent Gapdh Control Reagents VIC
were dissected at the indicated time points. Following fixation with Probe, Applied Biosystems) on an ABI Prism 7000 (Applied Biosystems)
methacarn solution for 4 hours, tissues were hydrated, stained with according to the manufacturer’s instructions.
Carmine Alum, and cleared with xylene. After analysis, the tissues were
processed for paraffin sectioning and stained with Hematoxylin and Eosin Mammary cell preparation, cell sorting and cell culture
(H&E). Inguinal mammary glands were dissected from 10-week-old virgin females
or pregnant mice at gestation day 10, mechanically disaggregated, and
Immunohistochemistry and immunofluorescence digested with collagenase (Sigma) and trypsin (Sigma) for 1 hour at 37°C
Immunohistochemistry was performed on methacarn-fixed or 4% (Sleeman et al., 2006). The resulting organoids were processed to single-
paraformaldehyde (PFA)-fixed, paraffin-embedded tissue sections using the cell suspensions by digestion with HyQTase (HyClone) for 10-15 minutes
following antibodies: Ki67 (Lab Vision), rabbit anti-milk serum (Marte et at 37°C and filtered through a 40-m cell strainer (Falcon). Cells were
al., 1995), pStat5 (Cell Signaling Technology), Stat5 (Santa Cruz stained as previously described (Sleeman et al., 2006) with the following
Biotechnology), estrogen receptor (ER; Esr1) (Santa Cruz Biotechnology) antibodies: FITC-CD24, PE-CD49f (Itga6), PE-Cy7-CD45 (Ptprc)
and progesterone receptor (PR; Pgr) (Thermo Scientific). (Pharmingen), APC-Sca1 (Ly6a), biotinylated-CD61 (Itgb3) (Biolegend)
Immunohistochemistry was carried out with the Discovery XT Staining and streptavidin-PE-Cy5.5 (eBioscience). FACS analysis and cell sorting
Module (Ventana Medical Systems), except for ER and PR were carried out on a MoFlo cell sorter (Beckman Coulter).
immunohistochemistry, which were performed manually. All sections were Colony-forming assays were performed by plating freshly sorted cells
counterstained with Hematoxylin (J.T.Baker). Quantification of pStat5, PR (500 cells) on irradiated 3T3-L1 feeder cells in Multiwell BD Primaria
and ER was performed by counting cells from at least 20 fields at a plates for 7 days in DMEM/Ham’s F12 mix (Invitrogen) with 10% fetal
magnification of 20⫻ and at least 2000 nuclei per sample. The number of calf serum, 100 IU/ml penicillin, 100 g/ml streptomycin (Invitrogen), 5
positive cells was expressed as a percentage of the total number of g/ml bovine pancreatic insulin (Sigma, cell culture tested solution) and
Hematoxylin-stained cells. Quantification of epithelial density and 10 ng/ml cholera toxin (Sigma). Aldefluor assay was performed according
proliferation index were performed on mammary gland sections stained to the manufacturer’s instructions (Stemcell Technologies).
with periodic acid Schiff (Ventana Medical Systems) and Hematoxylin, and
scanned with Miramax Scan (Carl Zeiss). For epithelial density, the area Hormone treatment
covered by epithelial cells (excluding lumen and blood vessels) was Six-week-old female mice were ovariectomized and treated 10 days later
measured and the ratio of epithelial area over total organ area was every 24 hours by subcutaneous injection of 17-estradiol (Sigma; 4 ng/g
calculated using Definiens software as described (Stoelzle et al., 2009). The body weight) in corn oil (Sigma) and sacrificed 48 hours later. For
same protocol was followed for the proliferation index using the area treatment with estrogen and progesterone, ovariectomized mice were
covered by Ki67-positive epithelial cells over total area of epithelial cells. injected with 17-estradiol and 48 hours later injected with 17-estradiol
At least three mice per genotype were scanned for each developmental plus progesterone (Sigma; 100 g/g body weight) daily for 72 hours.
stage.
Immunofluorescence was performed on 4% PFA-fixed, paraffin- Chemicals
embedded tissue sections stained with Rankl; tissues were then incubated NVP-BSK805 (Novartis, Switzerland) was freshly prepared in NMP/PEG
with Alexa Fluor 546 anti-goat IgG (Molecular Probes, Invitrogen), stained 300/Solutol HS15 (5%/80%/15%). Twelve-week-old mice were treated
with DAPI (Boehringer Mannheim), mounted in ProLong Gold antifade every 24 hours by oral gavage (120 mg/kg body weight) for 5 consecutive
reagent (Invitrogen), and analyzed with an LSM 700 scanning head and days. Glands were collected and fixed 4 hours after the final treatment.
Zen 2010 software (Carl Zeiss).
Microarray analysis
Crystal Violet staining was performed on cells grown in 24-well BD
RNA was isolated from three biological replicates per condition using
Primaria plates (BD Biosciences). The numbers and sizes of Crystal Violet-
the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s
stained colonies per well were quantified using ImagePro software (Media
Cybernetics). Three wells per genotype were examined in four independent instructions. RNA concentration was measured using a NanoDrop 1000
DEVELOPMENT
experiments. and the quality of the RNA assessed using the Agilent 2100 bioanalyzer
and RNA Nano Chip. Aliquots (100 ng) of extracted total RNA were
Immunoblotting amplified using the Ambion WT Expression Kit and the resulting sense-
Protein lysates were extracted from inguinal mammary glands using strand cDNA was fragmented and labeled using the Affymetrix
RIPA buffer [50 mM Tris pH 7.5, 1% Triton X-100, 150 mM NaCl, GeneChip WT Terminal Labeling Kit. Affymetrix GeneChip arrays were
0.5% sodium deoxycholate, 0.1% SDS, 5 mM EGTA, 10 mM NaF, hybridized following the GeneChip Whole Transcript (WT) Sense Target
2 mM sodium orthovanadate, 2 mM PMSF and protease inhibitor Labeling Assay Manual (Affymetrix) with a hybridization time of 16
cocktail (Pierce)]. Proteins (50 g) were resolved on SDS-PAGE (Bio- hours. The Affymetrix Fluidics protocol FS450_0007 was used for
Rad) and transferred to a PVDF membrane (Immobilon-FL, Millipore). washing. Scanning was performed with Affymetrix GCC Scan Control
PTP1B constrains mammary gland differentiation RESEARCH ARTICLE 119
Statistical analysis
Statistical significance was determined by two-tailed Student’s t-test. For
FACS analysis, a paired two-tailed Student’s t-test was performed.
RESULTS
Ptp1b deletion accelerates alveologenesis
Tyrosine phosphorylation plays an important role in mammary
gland alveologenesis. To determine whether PTP1B regulates this
process, we analyzed mammary glands of PTP1B-deficient and
wild-type (WT) female mice at different developmental stages.
Whole-mounts and histological analysis showed significant
changes in the structure of PTP1B-depleted compared with control
glands (Fig. 1A). In nulliparous mice at estrus, H&E and Ki67
staining revealed a twofold increase in epithelial cell density and
threefold more Ki67-positive cells in glands lacking PTP1B than
in WT glands (Fig. 1B-D). During early stages of pregnancy,
Ptp1b–/– glands showed an overall increase in the number of
epithelial cells and alveolar structures (Fig. 1B,C). These results
demonstrate that PTP1B constrains cell proliferation and
alveologenesis during estrus and early pregnancy.
cells and the number of colonies quantified. Ptp1b–/– MECs formed progenitor cells in human and mouse mammary tissues (Ginestier
approximately twice as many colonies as Ptp1b+/+ MECs, which et al., 2007; Cohn et al., 2010; Eirew et al., 2012). Using the
suggested an increase in progenitor cells in glands lacking PTP1B Aldefluor assay, we found a higher ALDH activity in MECs from
(Fig. 2B). Furthermore, the colonies formed by Ptp1b–/– MECs Ptp1b–/– than Ptp1b+/+ mice (supplementary material Fig. S1C),
were larger (Fig. 2B), consistent with our results showing increased further supporting an increase in the proportion of mammary
proliferation in glands from Ptp1b–/– compared with Ptp1b+/+ mice progenitor cells in PTP1B-deficient glands. Together, these results
(Fig. 1D). The increase in progenitor cell number was further tested show that PTP1B restrains the number of mammary progenitor
by FACS analysis for CD61, an epithelial progenitor marker cells in nulliparous mice.
120 RESEARCH ARTICLE Development 140 (1)
PTP1B negatively regulates ER activity might account for the increased number of MECs observed in
To investigate the molecular mediators of the observed increase glands from Ptp1b–/– mice.
in epithelial density and mammary progenitors in mammary We then tested whether overexpression of ER and/or estrogen
glands from Ptp1b–/– mice, we performed gene expression accounts for the increased transcription of ER targets in glands from
profiling of Ptp1b–/– and Ptp1b+/+ glands from mice at estrus. Ptp1b–/– mice, but found no difference in ER expression between
The absence of PTP1B increased the expression of several Ptp1b–/– and Ptp1b+/+ glands (Fig. 3B,C) and no difference in plasma
components of the cell cycle machinery: including cyclin B2, levels of estrogen between Ptp1b–/– and Ptp1b+/+ mice at estrus
cyclin A2, cyclin-dependent kinase 1 and topoisomerase 2A (supplementary material Fig. S3A). Further analysis showed no
(Fig. 3A; supplementary material Fig. S2A, Table S1). These differences in the plasma levels of progesterone in Ptp1b–/– and
results, combined with the increased proliferation observed by Ptp1b+/+ mice (supplementary material Fig. S3A). Thus, Ptp1b
immunohistochemistry (Fig. 1D), support a role for PTP1B in deletion appears to increase mammary cell proliferation by
the regulation of epithelial cell proliferation. enhancing the responsiveness of the mammary gland to normal
Further, analysis of the expression profiles of Ptp1b–/– and levels of circulating estrogen and progesterone. To test this
Ptp1b+/+ glands revealed increased expression of several estrogen- possibility directly, we assessed the effects of 17-estradiol treatment
DEVELOPMENT
responsive genes (supplementary material Fig. S2A): Pr, alone or in combination with progesterone on Ptp1b–/– and Ptp1b+/+
amphiregulin, Expi (Wfdc18), Egr2 and c-Myb. Furthermore, mice that were previously depleted of endogenous steroid hormones
quantitative RT-PCR and immunohistochemistry analysis revealed by ovariectomy. Treatment with 17-estradiol for 48 hours increased
an increase in PR expression in glands lacking PTP1B (Fig. 3B,C). the expression of PR in glands from Ptp1b–/– ovariectomized mice
Similarly, we found increased expression of Rankl, an established compared with ovariectomized WT littermates (Fig. 3D). These data
downstream target of PR (Fata et al., 2000; Beleut et al., 2010), in show that Ptp1b deletion increases ER activity in nulliparous glands.
glands deficient in PTP1B (Fig. 3B; supplementary material Fig. Furthermore, treatment of Ptp1b–/– and Ptp1b+/+ mice with 17-
S2B). These data suggest that PR-induced expression of Rankl estradiol and progesterone for 72 hours resulted in enhanced
PTP1B constrains mammary gland differentiation RESEARCH ARTICLE 121
compared with WT (Fig. 3E; supplementary material Fig. S2B). Stat5 (pStat5) and found a dramatic increase in pStat5 in the
Thus, PTP1B restrains epithelial cell proliferation by negatively absence of PTP1B (Fig. 4A). We then tested whether Jak2/Stat5
regulating ER activity and PR expression. inhibition blocks the increased epithelial cell proliferation
observed in Ptp1b–/– glands. Treatment of Ptp1b–/– and Ptp1b+/+
PTP1B depletion increases Stat5 phosphorylation mice with NVP-BSK805 [a selective Jak2 inhibitor that results
Genetic depletion of Stat5 revealed that this transcription factor in Stat5 dephosphorylation (Baffert et al., 2010)] inhibited Stat5
enhances the proliferation of epithelial cells in response to phosphorylation and, notably, significantly reduced proliferation
estrogen and progesterone stimuli, increases the number of in Ptp1b–/– glands (Fig. 4B).
122 RESEARCH ARTICLE Development 140 (1)
We next investigated whether overexpression of Prl and/or Prl- subpopulations (Fig. 5D). Furthermore, histological analysis
R or hyperphosphorylation of Jak2 accounts for the increased revealed that the alveolar structures in Ptp1b–/– but not Ptp1b+/+
pStat5 in glands from Ptp1b–/– mice. We found no difference in the glands were precociously distended, displayed lipid droplets and
plasma levels of Prl, in Prl-R expression or in Jak2 expression expressed milk proteins, which are all characteristics of
and phosphorylation between Ptp1b–/– and Ptp1b+/+ glands differentiated alveoli (Fig. 5A,B, Fig. 6A). Given the higher
(supplementary material Fig. S3A-D). This suggests that PTP1B number of alveoli in glands lacking PTP1B, the observed increase
acts via the Stat5 pathway in constraining epithelial cell in milk protein expression might be caused by enhanced expression
proliferation. and/or by an increase in the number of milk-producing cells. To
distinguish these possibilities, we assessed expression of the genes
PTP1B depletion accelerates mammary gland encoding the early pregnancy milk protein -casein and the late
differentiation during pregnancy pregnancy milk protein whey acidic protein (Wap), normalized to
We next assessed the consequences of Ptp1b deletion on mammary the expression of epithelial markers cytokeratin 8 and 18 in glands
gland development at later stages of pregnancy. Similar to the from control and Ptp1b–/– mice. PTP1B-depleted alveoli not only
phenotype at pregnancy day 3 (Fig. 1A,B), whole-mounts and expressed milk proteins precociously but also expressed a higher
H&E staining of glands showed that the absence of PTP1B also level of milk proteins per cell. PTP1B depletion resulted in 5.5-fold
results in the increased formation of alveolar structures at and 4.9-fold increases in the levels of -casein and Wap,
pregnancy days 7 and 10 (Fig. 5A-C). respectively (Fig. 6B; data not shown).
FACS analysis of MECs isolated from Ptp1b+/+ and Ptp1b–/– Next, we assessed the molecular mechanism underlying the
mice at pregnancy day 10 showed an increase in the luminal precocious lactogenesis seen in Ptp1b–/– glands. Immunoblotting
CD24hi Sca1– population, which is enriched in milk-expressing revealed increased phosphorylation of Stat5, a well-established
cells (Sleeman et al., 2006). No changes were observed in the other inducer of milk protein expression during pregnancy (Wakao et al.,
DEVELOPMENT
PTP1B constrains mammary gland differentiation RESEARCH ARTICLE 123
1994; Liu et al., 1997), in PTP1B-deficient glands compared with the tyrosine phosphatase PTP1B constrains these important
controls (Fig. 6C). To exclude the possibility that the observed processes. Alveologenesis is a developmental program
changes in pStat5 were due to changes in the stroma and not in characterized by the expansion and differentiation of mammary
epithelial cells, we performed immunostaining against pStat5. We progenitor cells into alveolar cells. Loss of PTP1B increases the
found that pStat5 in epithelial cells of glands lacking PTP1B was number of progenitor cells in nulliparous mice. This enhances the
markedly increased (Fig. 6D). We then tested whether Jak2 pool of cells able to generate alveolar structures and, thus, results
expression and/or phosphorylation is altered in glands lacking PTP1B in the increased alveolar density observed in Ptp1b–/– glands during
and found no difference in pJak2 between Ptp1b–/– and Ptp1b+/+ early pregnancy.
glands at pregnancy day 10 (supplementary material Fig. S3C,D). Several factors influence mammary gland alveologenesis.
To investigate whether Ptp1b deletion affects involution, we Mechanistically, we found that lack of PTP1B induces the
analyzed glands from Ptp1b–/– and Ptp1b+/+ mice 5 days after expression of several estrogen-responsive genes in nulliparous
cessation of suckling and observed no differences (supplementary glands, including Pr and its downstream target Rankl. PR plays a
material Fig. S3E). key role in epithelial cell proliferation and alveolar formation
DEVELOPMENT
These data show that PTP1B depletion precociously increases during early pregnancy (Lydon et al., 1995; Brisken et al., 1998;
Stat5 phosphorylation, thus triggering the expression of milk Mulac-Jericevic et al., 2003; Obr and Edwards, 2012). Therefore,
proteins. This suggests that PTP1B expression constrains the precocious alveolar development observed in Ptp1b–/– glands
lactogenesis during pregnancy. might be mediated by the overexpression and activation of PR,
which then precociously initiates alveologenesis.
DISCUSSION Estrogen and progesterone have been shown to regulate the
Tight regulation of mammary alveologenesis and lactogenesis is number and/or activity of MaSCs via a paracrine mechanism
fundamental for lactating species. In this study, we have shown that involving the Rank and Wnt pathways (Asselin-Labat et al., 2010;
124 RESEARCH ARTICLE Development 140 (1)
Joshi et al., 2010; Axlund and Sartorius, 2012). In glands lacking of Ptp1b delays or prevents mammary tumor formation in MMTV-
PTP1B, we observed an increase in ER and PR activity associated NeuNT and MMTV-NDL1 mice (Bentires-Alj and Neel, 2007;
with an increase in the number and activity of progenitor cells but Julien et al., 2007; Balavenkatraman et al., 2011). An exploration
not of MaSCs. The discrepancy between our results and those of this possibility is now warranted.
reported previously might be due to differences in the degrees of
ER and PR activation in the two models. Acknowledgements
We thank J. Regan (Institute of Cancer Research, Breakthrough Breast Cancer
In addition to increasing PR expression, PTP1B depletion Research, UK) for help with the MEC isolation and FACS sorting; B. Neel
increased the phosphorylation of Stat5, a key regulator of (BIDMC/Harvard Medical School, Ontario Cancer Institute) for providing Ptp1b
mammary luminal progenitor cells and alveologenesis (Yamaji et knockout mice; T. Radimerski and C. Pissot-Soldermann (NIBR) for supplying
al., 2009), suggesting that PTP1B restrains the number of NVP-BSK805; A. Doelemeyer (NIBR) for quantification of mammary epithelial
density; S. Bichet (FMI) for immunohistochemistry; T. Rolof (FMI) for microarray
mammary progenitor cells and regulates alveologenesis via Stat5
analysis; and S. Sarret (FMI) as well as further members of the M.B.-A.
dephosphorylation. Stat5 has been shown to promote the laboratory for advice and discussions.
proliferation of epithelial cells in response to estrogen and
progesterone stimuli, which indicated that they act in a common Funding
pathway (Miyoshi et al., 2001; Cui et al., 2004). Conceivably, Research in the laboratory of M.B.-A. is supported by the Novartis Research
Foundation and the European Research Council [ERC Starting Grant 243211-
PTP1B depletion increases ER activity and PR expression, which PTPsBDC].
in turn activates Stat5 and leads to increased alveologenesis. In
vitro studies have demonstrated that PTP1B can directly Competing interests statement
dephosphorylate Stat5 (Myers et al., 2001), raising an alternative The authors declare no competing financial interests.
possibility that lack of PTP1B independently increases PR
Supplementary material
expression via ER and Stat5 phosphorylation. These two Supplementary material available online at
possibilities are not mutually exclusive. http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.082941/-/DC1
But how does lack of PTP1B increase ER activity? In vitro
studies have shown that PTP1B dephosphorylates ER at tyrosine References
Aoki, N. and Matsuda, T. (2002). A nuclear protein tyrosine phosphatase TC-PTP
537, a residue known to inhibit estrogen binding and to reduce is a potential negative regulator of the PRL-mediated signaling pathway:
transcriptional activity of ER when phosphorylated (Arnold dephosphorylation and deactivation of signal transducer and activator of
et al., 1997). These data raise the possibility that PTP1B activates transcription 5a and 5b by TC-PTP in nucleus. Mol. Endocrinol. 16, 58-69.
Arnold, S. F., Melamed, M., Vorojeikina, D. P., Notides, A. C. and Sasson, S.
the estrogen pathway by regulating the phosphorylation of ER. (1997). Estradiol-binding mechanism and binding capacity of the human
Our results also support a role for PTP1B in lactogenesis. estrogen receptor is regulated by tyrosine phosphorylation. Mol. Endocrinol. 11,
PTP1B depletion induces the precocious expression of milk 48-53.
proteins due to an increase in Stat5 phosphorylation. Indeed, Stat5 Asselin-Labat, M. L., Sutherland, K. D., Barker, H., Thomas, R., Shackleton,
M., Forrest, N. C., Hartley, L., Robb, L., Grosveld, F. G., van der Wees, J. et
is a well-established regulator of lactogenesis, mediating Prl- al. (2007). Gata-3 is an essential regulator of mammary-gland morphogenesis
induced milk expression (Wakao et al., 1994; Liu et al., 1997). and luminal-cell differentiation. Nat. Cell Biol. 9, 201-209.
Taken together, our results suggest a role for PTP1B as a temporal Asselin-Labat, M. L., Vaillant, F., Sheridan, J. M., Pal, B., Wu, D., Simpson, E.
regulator of mammary gland development that downregulates the R., Yasuda, H., Smyth, G. K., Martin, T. J., Lindeman, G. J. et al. (2010).
Control of mammary stem cell function by steroid hormone signalling. Nature
progesterone and Stat5 pathway(s) and thus prevents the 465, 798-802.
inappropriate onset of alveologenesis and lactogenesis during Axlund, S. D. and Sartorius, C. A. (2012). Progesterone regulation of stem and
pregnancy. progenitor cells in normal and malignant breast. Mol. Cell. Endocrinol. 357, 71-
79.
Mammary gland development and differentiation are regulated Baffert, F., Régnier, C. H., De Pover, A., Pissot-Soldermann, C., Tavares, G. A.,
by a complex mechanism involving several different pathways Blasco, F., Brueggen, J., Chène, P., Drueckes, P., Erdmann, D. et al. (2010).
(Hennighausen and Robinson, 2005; Brisken and O’Malley, 2010). Potent and selective inhibition of polycythemia by the quinoxaline JAK2 inhibitor
We cannot exclude the possibility that PTP1B acts via other NVP-BSK805. Mol. Cancer Ther. 9, 1945-1955.
Balavenkatraman, K. K., Aceto, N., Britschgi, A., Mueller, U., Bence, K. K.,
pathways in constraining mammary gland alveologenesis and Neel, B. G. and Bentires-Alj, M. (2011). Epithelial protein-tyrosine
lactogenesis. For example, PTP1B is a well-known regulator of the phosphatase 1B contributes to the induction of mammary tumors by HER2/Neu
insulin and leptin pathways in other organs, and mice lacking but is not essential for tumor maintenance. Mol. Cancer Res. 9, 1377-1384.
Beleut, M., Rajaram, R. D., Caikovski, M., Ayyanan, A., Germano, D., Choi,
PTP1B are insulin and leptin hypersensitive (Elchebly et al., 1999; Y., Schneider, P. and Brisken, C. (2010). Two distinct mechanisms underlie
Klaman et al., 2000). In the light of reports of a role for insulin, progesterone-induced proliferation in the mammary gland. Proc. Natl. Acad. Sci.
Igf1 and Igf2 in mammary gland morphogenesis (Kleinberg et al., USA 107, 2989-2994.
2000; Brisken et al., 2002; Hovey et al., 2003; Berlato and Doppler, Bentires-Alj, M. and Neel, B. G. (2007). Protein-tyrosine phosphatase 1B is
required for HER2/Neu-induced breast cancer. Cancer Res. 67, 2420-2424.
2009), PTP1B might also regulate mammary gland morphogenesis Berlato, C. and Doppler, W. (2009). Selective response to insulin versus insulin-
via its inhibitory effects on these pathways. like growth factor-I and -II and up-regulation of insulin receptor splice variant B
Epidemiological studies have shown that early menarche, late in the differentiated mouse mammary epithelium. Endocrinology 150, 2924-
2933.
menopause and late age of first pregnancy are all risk factors for Brisken, C. (2002). Hormonal control of alveolar development and its implications
developing sporadic breast cancer (Medina, 2005). Conversely, for breast carcinogenesis. J. Mammary Gland Biol. Neoplasia 7, 39-48.
DEVELOPMENT
early full-term pregnancy (<24 years) decreases lifetime breast Brisken, C. and O’Malley, B. (2010). Hormone action in the mammary gland.
cancer risk. Clearly, the hormonal milieu and breast development Cold Spring Harb. Perspect. Biol. 2, a003178.
Brisken, C. and Rajaram, R. D. (2006). Alveolar and lactogenic differentiation. J.
cycles, possibly through changes in the differentiation state of Mammary Gland Biol. Neoplasia 11, 239-248.
breast stem/progenitor cells, affect the susceptibility of the breast Brisken, C., Park, S., Vass, T., Lydon, J. P., O’Malley, B. W. and Weinberg, R.
to oncogenic transformation. Our finding that Ptp1b deletion A. (1998). A paracrine role for the epithelial progesterone receptor in mammary
induces precocious differentiation of the mammary gland raises the gland development. Proc. Natl. Acad. Sci. USA 95, 5076-5081.
Brisken, C., Ayyannan, A., Nguyen, C., Heineman, A., Reinhardt, F., Tan, J.,
possibility that the cells of origin of Her2-evoked mammary tumors Dey, S. K., Dotto, G. P. and Weinberg, R. A. (2002). IGF-2 is a mediator of
are decreased in Ptp1b–/– mice. This would explain why deletion prolactin-induced morphogenesis in the breast. Dev. Cell 3, 877-887.
PTP1B constrains mammary gland differentiation RESEARCH ARTICLE 125
Bruno, R. D. and Smith, G. H. (2011). Functional characterization of stem cell Lydon, J. P., DeMayo, F. J., Funk, C. R., Mani, S. K., Hughes, A. R.,
activity in the mouse mammary gland. Stem Cell Rev. 7, 238-247. Montgomery, C. A., Jr, Shyamala, G., Conneely, O. M. and O’Malley, B. W.
Cohn, E., Ossowski, L., Bertran, S., Marzan, C. and Farias, E. F. (2010). (1995). Mice lacking progesterone receptor exhibit pleiotropic reproductive
RARalpha1 control of mammary gland ductal morphogenesis and wnt1- abnormalities. Genes Dev. 9, 2266-2278.
tumorigenesis. Breast Cancer Res. 12, R79. Marte, B. M., Jeschke, M., Graus-Porta, D., Taverna, D., Hofer, P., Groner, B.,
Cui, Y., Riedlinger, G., Miyoshi, K., Tang, W., Li, C., Deng, C. X., Robinson, G. Yarden, Y. and Hynes, N. E. (1995). Neu differentiation factor/heregulin
W. and Hennighausen, L. (2004). Inactivation of Stat5 in mouse mammary modulates growth and differentiation of HC11 mammary epithelial cells. Mol.
epithelium during pregnancy reveals distinct functions in cell proliferation, Endocrinol. 9, 14-23.
survival, and differentiation. Mol. Cell. Biol. 24, 8037-8047. Medina, D. (2005). Mammary developmental fate and breast cancer risk. Endocr.
Deome, K. B., Faulkin, L. J., Jr, Bern, H. A. and Blair, P. B. (1959). Development Relat. Cancer 12, 483-495.
of mammary tumors from hyperplastic alveolar nodules transplanted into gland- Miyoshi, K., Shillingford, J. M., Smith, G. H., Grimm, S. L., Wagner, K. U.,
free mammary fat pads of female C3H mice. Cancer Res. 19, 515-520. Oka, T., Rosen, J. M., Robinson, G. W. and Hennighausen, L. (2001). Signal
Eirew, P., Kannan, N., Knapp, D. J., Vaillant, F., Emerman, J. T., Lindeman, G. transducer and activator of transcription (Stat) 5 controls the proliferation and
J., Visvader, J. E. and Eaves, C. J. (2012). Aldehyde dehydrogenase activity is a differentiation of mammary alveolar epithelium. J. Cell Biol. 155, 531-542.
biomarker of primitive normal human mammary luminal cells. Stem Cells 30, Mulac-Jericevic, B., Lydon, J. P., DeMayo, F. J. and Conneely, O. M. (2003).
344-348. Defective mammary gland morphogenesis in mice lacking the progesterone
Elchebly, M., Payette, P., Michaliszyn, E., Cromlish, W., Collins, S., Loy, A. L., receptor B isoform. Proc. Natl. Acad. Sci. USA 100, 9744-9749.
Normandin, D., Cheng, A., Himms-Hagen, J., Chan, C. C. et al. (1999). Myers, M. P., Andersen, J. N., Cheng, A., Tremblay, M. L., Horvath, C. M.,
Increased insulin sensitivity and obesity resistance in mice lacking the protein Parisien, J. P., Salmeen, A., Barford, D. and Tonks, N. K. (2001). TYK2 and
tyrosine phosphatase-1B gene. Science 283, 1544-1548. JAK2 are substrates of protein-tyrosine phosphatase 1B. J. Biol. Chem. 276,
Fata, J. E., Kong, Y. Y., Li, J., Sasaki, T., Irie-Sasaki, J., Moorehead, R. A., 47771-47774.
Elliott, R., Scully, S., Voura, E. B., Lacey, D. L. et al. (2000). The osteoclast Neville, M. C., McFadden, T. B. and Forsyth, I. (2002). Hormonal regulation of
differentiation factor osteoprotegerin-ligand is essential for mammary gland mammary differentiation and milk secretion. J. Mammary Gland Biol. Neoplasia
development. Cell 103, 41-50. 7, 49-66.
Faulkin, L. J., Jr and Deome, K. B. (1960). Regulation of growth and spacing of Obr, A. E. and Edwards, D. P. (2012). The biology of progesterone receptor in
gland elements in the mammary fat pad of the C3H mouse. J. Natl. Cancer Inst. the normal mammary gland and in breast cancer. Mol. Cell. Endocrinol. 357,
24, 953-969. 4-17.
Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, Regan, J. L., Kendrick, H., Magnay, F. A., Vafaizadeh, V., Groner, B. and
S., Ellis, B., Gautier, L., Ge, Y., Gentry, J. et al. (2004). Bioconductor: open
Smalley, M. J. (2011). c-Kit is required for growth and survival of the cells of
software development for computational biology and bioinformatics. Genome
origin of Brca1-mutation-associated breast cancer. Oncogene 31, 869-883.
Biol. 5, R80.
Shackleton, M., Vaillant, F., Simpson, K. J., Stingl, J., Smyth, G. K., Asselin-
Ginestier, C., Hur, M. H., Charafe-Jauffret, E., Monville, F., Dutcher, J.,
Labat, M. L., Wu, L., Lindeman, G. J. and Visvader, J. E. (2006). Generation
Brown, M., Jacquemier, J., Viens, P., Kleer, C. G., Liu, S. et al. (2007). ALDH1
of a functional mammary gland from a single stem cell. Nature 439, 84-88.
is a marker of normal and malignant human mammary stem cells and a
Sleeman, K. E., Kendrick, H., Ashworth, A., Isacke, C. M. and Smalley, M. J.
predictor of poor clinical outcome. Cell Stem Cell 1, 555-567.
(2006). CD24 staining of mouse mammary gland cells defines luminal epithelial,
Gouilleux, F., Wakao, H., Mundt, M. and Groner, B. (1994). Prolactin induces
myoepithelial/basal and non-epithelial cells. Breast Cancer Res. 8, R7.
phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and
induction of transcription. EMBO J. 13, 4361-4369. Sleeman, K. E., Kendrick, H., Robertson, D., Isacke, C. M., Ashworth, A. and
Groner, B. (2002). Transcription factor regulation in mammary epithelial cells. Smalley, M. J. (2007). Dissociation of estrogen receptor expression and in vivo
Domest. Anim. Endocrinol. 23, 25-32. stem cell activity in the mammary gland. J. Cell Biol. 176, 19-26.
Hennighausen, L. and Robinson, G. W. (2005). Information networks in the Stingl, J., Eirew, P., Ricketson, I., Shackleton, M., Vaillant, F., Choi, D., Li, H. I.
mammary gland. Nat. Rev. Mol. Cell Biol. 6, 715-725. and Eaves, C. J. (2006a). Purification and unique properties of mammary
Hovey, R. C., Harris, J., Hadsell, D. L., Lee, A. V., Ormandy, C. J. and epithelial stem cells. Nature 439, 993-997.
Vonderhaar, B. K. (2003). Local insulin-like growth factor-II mediates prolactin- Stingl, J., Raouf, A., Eirew, P. and Eaves, C. J. (2006b). Deciphering the
induced mammary gland development. Mol. Endocrinol. 17, 460-471. mammary epithelial cell hierarchy. Cell Cycle 5, 1519-1522.
Joshi, P. A., Jackson, H. W., Beristain, A. G., Di Grappa, M. A., Mote, P. A., Stoelzle, T., Schwarb, P., Trumpp, A. and Hynes, N. E. (2009). c-Myc affects
Clarke, C. L., Stingl, J., Waterhouse, P. D. and Khokha, R. (2010). mRNA translation, cell proliferation and progenitor cell function in the mammary
Progesterone induces adult mammary stem cell expansion. Nature 465, 803-807. gland. BMC Biol. 7, 63.
Julien, S. G., Dubé, N., Read, M., Penney, J., Paquet, M., Han, Y., Kennedy, B. Van Keymeulen, A., Rocha, A. S., Ousset, M., Beck, B., Bouvencourt, G.,
P., Muller, W. J. and Tremblay, M. L. (2007). Protein tyrosine phosphatase 1B Rock, J., Sharma, N., Dekoninck, S. and Blanpain, C. (2011). Distinct stem
deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and cells contribute to mammary gland development and maintenance. Nature 479,
protects from lung metastasis. Nat. Genet. 39, 338-346. 189-193.
Klaman, L. D., Boss, O., Peroni, O. D., Kim, J. K., Martino, J. L., Zabolotny, J. Visvader, J. E. (2009). Keeping abreast of the mammary epithelial hierarchy and
M., Moghal, N., Lubkin, M., Kim, Y. B., Sharpe, A. H. et al. (2000). Increased breast tumorigenesis. Genes Dev. 23, 2563-2577.
energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in Visvader, J. E. and Smith, G. H. (2011). Murine mammary epithelial stem cells:
protein-tyrosine phosphatase 1B-deficient mice. Mol. Cell. Biol. 20, 5479-5489. discovery, function, and current status. Cold Spring Harb. Perspect. Biol. 3.
Kleinberg, D. L., Feldman, M. and Ruan, W. (2000). IGF-I: an essential factor in Wakao, H., Gouilleux, F. and Groner, B. (1994). Mammary gland factor (MGF) is
terminal end bud formation and ductal morphogenesis. J. Mammary Gland Biol. a novel member of the cytokine regulated transcription factor gene family and
Neoplasia 5, 7-17. confers the prolactin response. EMBO J. 13, 2182-2191.
Kordon, E. C. and Smith, G. H. (1998). An entire functional mammary gland may Wartmann, M., Cella, N., Hofer, P., Groner, B., Liu, X., Hennighausen, L. and
comprise the progeny from a single cell. Development 125, 1921-1930. Hynes, N. E. (1996). Lactogenic hormone activation of Stat5 and transcription
LaBarge, M. A., Petersen, O. W. and Bissell, M. J. (2007). Of of the beta-casein gene in mammary epithelial cells is independent of p42 ERK2
microenvironments and mammary stem cells. Stem Cell Rev. 3, 137-146. mitogen-activated protein kinase activity. J. Biol. Chem. 271, 31863-31868.
Liu, X., Robinson, G. W., Wagner, K. U., Garrett, L., Wynshaw-Boris, A. and Yamaji, D., Na, R., Feuermann, Y., Pechhold, S., Chen, W., Robinson, G. W.
Hennighausen, L. (1997). Stat5a is mandatory for adult mammary gland and Hennighausen, L. (2009). Development of mammary luminal progenitor
development and lactogenesis. Genes Dev. 11, 179-186. cells is controlled by the transcription factor STAT5A. Genes Dev. 23, 2382-2387.
DEVELOPMENT